The Aspergillus giganteus antifungal protein AFPNN5353 activates the cell wall integrity pathway and perturbs calcium homeostasis

Background: The antifungal protein AFP(NN5353) is a defensin-like protein of Aspergillus giganteus. It belongs to a group of secretory proteins with low molecular mass, cationic character and a high content of cysteine residues. The protein inhibits the germination and growth of filamentous ascomycetes, including important human and plant pathogens and the model organsims Aspergillus nidulans and Aspergillus niger. Results: We determined an AFP(NN5353) hypersensitive phenotype of non-functional A. nidulans mutants in the protein kinase C (Pkc)/mitogen-activated protein kinase (Mpk) signalling pathway and the induction of the alpha-glucan synthase A (agsA) promoter in a transgenic A. niger strain which point at the activation of the cell wall integrity pathway (CWIP) and the remodelling of the cell wall in response to AFP(NN5353). The activation of the CWIP by AFP(NN5353), however, operates independently from RhoA which is the central regulator of CWIP signal transduction in fungi. Furthermore, we provide evidence that calcium (Ca2+) signalling plays an important role in the mechanistic function of this antifungal protein. AFP(NN5353) increased about 2-fold the cytosolic free Ca2+ ([Ca2+](c)) of a transgenic A. niger strain expressing codon optimized aequorin. Supplementation of the growth medium with CaCl2 counteracted AFP(NN5353) toxicity, ameliorated the perturbation of the [Ca2+](c) resting level and prevented protein uptake into Aspergillus sp. cells. Conclusions: The present study contributes new insights into the molecular mechanisms of action of the A. giganteus antifungal protein AFP(NN5353). We identified its antifungal activity, initiated the investigation of pathways that determine protein toxicity, namely the CWIP and the Ca2+ signalling cascade, and studied in detail the cellular uptake mechanism in sensitive target fungi. This knowledge contributes to define new potential targets for the development of novel antifungal strategies to prevent and combat infections of filamentous fungi which have severe negative impact in medicine and agriculture.